Action of 6‐methyl Purine on Rna Synthesis: Incorporation of the Adenine Analogue Into Coding Sequences and Polyadenylate Tracts of Messenger Rna in Chlorella

Abstract

Summary 6‐methyl purine has been tritiated and, by two‐dimensional paper chromatography, an impurity and two degradation products formed during tritiation have been removed. The authentic 6‐methyl purine has been identified by n.m.r. spectroscopy. Chlorella 211‐8p grown autotrophically in the presence of 1 mM tritiated 6‐methyl purine incorporates radioactivity into RNA. Electrophoretic analysis shows that 45% of the incorporated radioactivity is in RNA fragments with less than 90 bases. There is only trace incorporation into 25s, 18s and 5s rRNA but tRNA accommodates 8% of incorporated label. The bulk of radioactivity found in completed RNA is present in a polydisperse size range of molecules which are identified as mRNA. They are recovered as poly(A)+ RNA by oligo (dT) cellulose binding and are of an appropriate size for a messenger function, since the message which contributes the greatest weight to the total population is of 0·65 × 106mol. wt and could accommodate 677 codons, while electrophoretic analysis of polypeptides from Chlorella shows the size which contributes the greatest weight to be 5 × 104 mol. wt, equivalent to 450 amino acids. This correlates well with the capacity of the predominant message, allowing for the presence of a poly(A) tract. 6‐methyl purine is incorporated without modification, since two‐dimensional thin layer chromatography of ribosides derived from mRNA showed that radioactivity co‐chromatographed exclusively with authentic 6‐methyl purine riboside. Poly(A) tracts, prepared from mRNA by digestion of the coding sequences, can be separated from the resulting mononucleotides by electrophoresis. Accurate location of the mononucleotides and the selection of authentic poly(A) segments with oligo (dT) cellulose establishes that tracts of 100 to 200 adenine residues contribute the bulk of poly(A) and so corrects an earlier report that tracts in Chlorella are unusually small. The established sizes of poly(A) tracts and complete messenger molecules indicate a predicted distribution of adenine bases between tract and coding sequences which is close to the observed values of 21 and 89% seen in digests of isolated mRNA. A similarity with poly(A) metabolism in mammalian cells is detected by analysis of the number of tracts of each size, which shows that the smaller sizes are numerically predominant. 6‐methyl purine is recovered from both poly(A) tract and coding sequences of mRNA after a 1 h period of autotrophic growth with the inhibitor. At the end of this period one in every three adenines in the tract sequences and one in every 10 adenines in the coding sequences has been substituted by 6‐methyl purine. The incidence in messengers being synthesized in the presence of 6‐methyl purine is probably higher since messengers persisting from before exposure to the inhibitor will give a lower average incidence in the total messenger population. Using minimum estimates of 6‐methyl purine substitution it is calculated that coding sequences for a protein of 40000 mol. wt have only a 1 in 5·3 × 1011pro...

ISOLATION OF POLYSOMES AND POLY(A)‐CONTAINING RNA FROM CHLORELLA FUSCA VAR. VACUOLATA

“…One possible cause of the broader size distribution in poly(A)-containing RNA from polysomes was that it resulted from the cycloheximide treatment used to prevent ribosome 'run oiT', but no efTect was detected when the size distribution of total RNA-derived poly(A)-containing RNA was compared from cells treated and untreated with the antibiotic (data not shown). The size distribution of poly(A)-tracts within poly(A)-containing RNA is shown in Figure 6, which conforms substantially with the data of Lambe and John (1979) correcting our earlier estimate . When corrected for the distribution of radioactivity with tract length the most abundant sizes are about 155 nucleotides and 50 nucleotides (Fig.…”

“…Size calibration was established from 5-8S, 5S and 4S RNA run on parallel gels. abundant size class of polypeptides in C. fusca has 455 amino acid residues per chain which requires only 0-44 x 10^ daltons (1365 nucleotides) of translated RNA sequence (Lambe and John, 1979). It should be noted that although the size distribution of undegraded poly(A)-containing RNA samples (Fig.…”

Use of E. coli Purine Nucleoside Phosphorylase in the Treatment of Solid Tumors

“…Because the carbon-carbon bond of MeP-R is quite different from that of a carbon-nitrogen bond of adenosine, incorporation of MeP-R into double stranded regions of RNA in place of adenosine would result in double stranded regions that could not be unfolded by ADAR. Conversely, since the 6- amino group of adenosine participates in one of the two hydrogen bonds formed when adenine undergoes Watson-Crick base pairing, it is possible that the 6 methyl group of MeP-R does not form hydrogen bonds with uracil, preventing formation of double stranded regions in the RNA in the first place [89, 90]. Either of these consequences related to incorporation of MeP into RNA could disrupt important metabolic processes and would be quite cytotoxic to the cell [81].…”